CN115006405A - Application of dihydromyricetin combined with ceftiofur hydrochloride in anti-MRSA infection - Google Patents

Abstract

The invention relates to the application of dihydromyricetin and ceftiofur hydrochloride in anti-MRSA drug-resistant bacterial infection. Therefore, the present invention provides a drug combination for anti-MRSA drug-resistant bacterial infection. The combination is dihydromyricetin and cephalosporin hydrochloride. The combination of thiifur, the present invention has verified through in vitro test that the combination of dihydromyricetin and ceftiofur hydrochloride has a good synergistic effect, and is effective against MRSA resistant standard strain ATCC 33591 and MRSA isolated strain. The invention provides a new idea for expanding the clinical application of dihydromyricetin and treating methicillin-resistant staphylococcus aureus (MRSA), and provides a new idea for the combined treatment of methicillin-resistant staphylococcus aureus (MRSA) infection and the expansion of dihydromyricetin The clinical application of myricetin provides reference.

Description

Application of dihydromyricetin combined with ceftiofur hydrochloride in anti-MRSA infection

technical field

The invention relates to a drug combination against MRSA drug-resistant bacteria, which belongs to the field of prevention and treatment of animal bacterial diseases.

Background technique

Menicillin resistant Staphylococcus aureus (MRSA) is a Gram-positive pathogen that causes clinical diseases such as mastitis, pneumonia, skin suppurative inflammation, ulcerative plantaritis, and bacteremia in animals. It is also one of the pathogens with the most serious drug resistance and the most difficult to cure in the world. Traditional antibacterial drugs can no longer solve the diseases caused by the drug-resistant bacteria. Finding effective antibacterial substances from natural drugs is an important direction for the development of new antibacterial drugs. The invention can enhance the sensitivity of MRSA resistant bacteria to ceftiofur hydrochloride, reduce the dosage of antibiotic ceftiofur hydrochloride, and provide an effective drug combination for the treatment of MRSA infection in veterinary clinics. Value.

Ceftiofu hydrochloride (Ceftiofu Hcl, CEF), also known as ceftiofu hydrochloride, is light yellow powder, insoluble in water, easily soluble in methanol, dimethyl sulfoxide, etc. Dosing by injection. Ceftiofur hydrochloride is a third-generation cephalosporin specially used for animals. It has a broad-spectrum bactericidal effect and inhibits both Gram-negative and positive bacteria. It is mainly used to treat infectious diseases of pigs, cattle, horses, dogs and one-day-old chicks caused by sensitive bacteria. In pigs, CEF resulted in a C(max) of 11.8 mg/mL and an AUC of 216 mgxh/mL 1 to 4 hours after intramuscular injection. In dairy cows, intramuscular injection of CEF at 2.2 mg/kg, once every 5 days, can effectively treat acute postpartum metritis in dairy cows.

Dihydromyricetin (DMY), also known as snake skin, belongs to flavonoids, widely exists in the genus Snake grape, and is easily soluble in hot water, hot ethanol and acetone. Dihydromyricetin has been shown to have multiple pharmacological effects including anti-inflammatory, antioxidant, antiviral, anticancer, antibacterial, neuroprotective, antibacterial, and lipid and glucose metabolism-modulating activities. Studies have shown that dihydromyricetin has excellent antibacterial and bactericidal properties against Staphylococcus aureus, and the minimum inhibitory concentration and minimum bactericidal concentration are 0.125 and 0.25 mg/mL, respectively.

SUMMARY OF THE INVENTION

The present invention has the following advantages:

The invention finds that the combined use of dihydromyricetin and ceftiofur hydrochloride enhances the efficacy of ceftiofur hydrochloride against MRSA, and can solve the problem of drug resistance of MRSA to ceftiofur hydrochloride.

Dihydromyricetin has good bacteriostatic effect, but its research on veterinary clinic is still relatively small.

In the present invention, dihydromyricetin is studied and used for treating MRSA drug-resistant bacterial infection.

In the present invention, dihydromyricetin and ceftiofur hydrochloride are used in combination, and it is found that the combination can improve the sensitivity of antibiotics, reduce the dosage of antibiotics, and has a synergistic combination. The present invention has been tested and verified the effect of this combination.

The pharmaceutical combination of the present invention is a combination of dihydromyricetin and ceftiofur hydrochloride, and the weight ratio of the two is 1-10:1. Preferably, the weight ratio of the two is 1-5:1. Particularly preferably, the weight ratio of the two is 1-2:1. Most preferably, the consumption of dihydromyricetin is twice that of ceftiofur hydrochloride, that is, the weight ratio of the two is 2:1.

The combination of the present invention comprises that dihydromyricetin and ceftiofur hydrochloride are mixed together to prepare a pharmaceutical composition, which is applied simultaneously during use, or dihydromyricetin and ceftiofur hydrochloride are separately prepared into pharmaceutical compositions, which are combined when used use. Wherein, in the pharmaceutical composition prepared by mixing the dihydromyricetin and ceftiofur hydrochloride together, other ingredients may also be included, and the two are separately prepared as pharmaceutical compositions, which are used in combination when used, and may also include a combination of Use other ingredients.

The pharmaceutical composition of the present invention is in the form of a pharmaceutical preparation, selected from oral preparations and non-oral preparations, wherein the non-oral preparations include injections. Preferably, in the pharmaceutical combination of the present invention, each dose of dihydromyricetin is about 2g, and each dose of ceftiofur hydrochloride is about 1g.

The invention opens up a new use for dihydromyricetin, can be used as a synergist for cephalosporin antibiotics, and also provides a safe and effective auxiliary drug for the treatment of MRSA infection in veterinary clinics and the drug resistance control of bacteria. Early technical support.

Therefore, the present invention provides an application of dihydromyricetin, which in combination with cephalosporin antibiotics can significantly reduce the minimum inhibitory concentration of the cephalosporin antibiotics against MRSA-resistant bacteria. The weight ratio of dihydromyricetin and cephalosporin antibiotics is 1-10:1, preferably, the weight ratio of the two is 1-5:1. Most preferably, the weight ratio of the two is 1-2:1. In the case of curative effect, the dosage of dihydromyricetin is twice that of ceftiofur hydrochloride.

The application of the dihydromyricetin of the present invention lies in that the dihydromyricetin has the effect of increasing the resistance of cephalosporin antibiotics to MRSA resistant bacteria infection. The cephalosporin antibiotic is preferably ceftiofur hydrochloride. When the weight ratio of dihydromyricetin and ceftiofur hydrochloride is 2:1, the two have a synergistic effect.

The present invention finds through experiments that the compound medicine obtained by combining dihydromyricetin and ceftiofur hydrochloride antibiotic in a weight ratio of 2:1 has the best therapeutic effect and has the strongest effect on MRSA strains.

The chessboard dilution method test proved that when dihydromyricetin and ceftiofur hydrochloride are used in combination, they can synergistically enhance the antibacterial activity of ceftiofur hydrochloride and reverse the resistance of MRSA isolates to ceftiofur hydrochloride. This greatly reduces the use of antibacterial drugs and has the potential to be an inhibitor of drug-resistant MRSA.

The beneficial effects of the present invention are described below through experimental data.

1. Test consumables

1. Reagent drugs

Dihydromyricetin: produced by Shanghai Yuanye Biological Co., Ltd.; content: 98%; batch number: RFS-E00211812016. Ceftiofur hydrochloride: produced by Shanghai Yuanye Biological Co., Ltd., content: 98%, batch number: H19N9Z75089. Dihydromyricetin was dissolved in DMSO to prepare 5120 μg/mL as a reserve mother solution, which was stored in a -20°C refrigerator for future use; ceftiofur hydrochloride was dissolved in DMSO to prepare 5120 μg/mL as a reserve mother solution, which was stored in a -20°C refrigerator for future use.

2. Strains

MRSA strain ATCC 33591: Provided by the Research Center of Traditional Chinese Veterinary Medicine, China Agricultural University. Staphylococcus aureus standard strain ATCC 29213: provided by the Research Center of Traditional Chinese Veterinary Medicine, China Agricultural University. MRSA strains from healthy pigs from a pig farm in Shandong: Provided by the Department of Veterinary Toxicology and Pharmacology, School of Veterinary Medicine, China Agricultural University. Staphylococcus aureus was activated by streaking on BHI medium, cultured at 37°C for 18h, and stored at 4°C for later use.

3. Culture medium

MHB broth medium: purchased from Qingdao Haibo Biotechnology Co., Ltd., accurately weigh 2.1g of MHB broth medium powder, pour it into a conical flask filled with 100mL of distilled water, shake to dissolve, cover with a sealing film and 121 ℃ After autoclaving for 20 min, store at 4°C for later use.

MHA agar medium: purchased from Qingdao Haibo Biotechnology Co., Ltd., accurately weigh 8.4g of MHA agar medium powder, pour it into a conical flask with 100 mL of distilled water, shake to dissolve, cover with a sealing film, and then press the pressure at 121 °C. After sterilization for 20 min, store at 4°C for later use.

BHI brain heart infusion agar medium: purchased from Qingdao Haibo Biotechnology Co., Ltd., accurately weigh 5.2g of BHI brain heart infusion agar medium powder, pour it into a conical flask containing 100 mL of distilled water, shake to dissolve, Cover with parafilm, autoclave at 121 °C for 20 min, and store at 4 °C for later use.

4. Instruments

Two-person single-sided vertical ultra-clean workbench (Suzhou Purification Equipment Co., Ltd.); automatic vertical pressure steam sterilizer (Zhiwei Instrument Co., Ltd.); electronic analytical balance (Sartorius); constant temperature shaking incubator, biochemical incubator (Shanghai No. 1 Hengke Instrument Co., Ltd.); portable bacterial turbidimeter (Shanghai Xinrui Instrument Co., Ltd.); electric heating constant temperature drying oven (Shanghai Jinghong Industrial Equipment Co., Ltd.); -80℃ ultra-low temperature refrigerator (Thermo Fisher Technology Co., Ltd. Company); micro-adjustable sampler (Eeppendorf).

2. Test method

1. Preparation of the test bacterial solution

Dissolve the Staphylococcus aureus standard strain ATCC 29213, MRSA standard strain ATCC33591 and isolated drug-resistant strains stored in glycerol at -80°C at room temperature, and inoculate the bacterial liquid on BHI agar by the three-zone streaking method in the ultra-clean bench. , in an incubator at 37°C for 24h of inversion, pick a typical single colony and inoculate it in MHB broth, incubate for 6-8h on a shaking table (37°C, 180r/min), and adjust the bacterial solution to 0.5 McFarland with normal saline Turbidity standard (concentration is about 10 ⁸ CFU/mL), the bacterial liquid was diluted 100 times with MHB broth to make the bacterial liquid concentration 10 ⁶ CFU/mL, as the test bacterial liquid.

2. Determination of single drug susceptibility MIC value

MRSA ATCC 33591 was used as the test strain, and ATCC 29213 was used as the quality control strain. The MIC determination of ceftiofur hydrochloride and dihydromyricetin was carried out by the micro-broth dilution method specified by CLSI.

3. Micro-checkerboard dilution method broth combined with drug sensitivity

In order to detect the antibacterial effect of the two active ingredients on MRSA after the combined application, the micro-checkerboard dilution method was used, that is, the combination of dihydromyricetin and ceftiofur hydrochloride. Staphylococcus aureus ATCC 29213 was used as the quality control strain, MRSA strain ATCC 33591 was used as the test strain, and drug-resistant strains were isolated as the verification strain.

Based on the MIC results, the micro-checkerboard dilution method uses a 96-well sterile microplate, and designs two drugs 4 times, 2 times, 1 times, 1/2 times, 1/4 times, 1/8 times, and 1/16 times. 8 × 8 combinations were performed at 1/32 times the MIC. The two drugs were respectively doubling dilution, the traditional Chinese medicine monomer was diluted in the horizontal column, 50 μL per well; the antibiotic drugs were added in the vertical column, 50 μL per well. After mixing the test bacterial solution, it was added to each well of a 96-well plate with an eight-well pipette, 100 μL per well. At the same time, the single MIC, negative and positive wells of dihydromyricetin and ceftiofur hydrochloride were set as controls. Incubate at 37°C, observe and record the results.

FICI index (graded inhibitory concentration index FICI) calculation and result judgment:

FICI index interpretation standard: when FICI≤0.5, it is synergistic effect; when 0.5<FICI≤1, it is additive effect; when 1<FICI≤2, it is irrelevant effect; when FICI>2, it is antagonistic effect.

3. Test results

The MIC value of the combined drug susceptibility was determined by taking the minimum concentration with no bacterial growth observed by the naked eye as the MIC of the drug. The MIC of ceftiofur hydrochloride against Staphylococcus aureus quality control bacteria ATCC 29213 was 1 μg/mL respectively, which met the quality control range of 0.25 to 1 μg. /mL.

The results of the combined drug susceptibility test are shown in Table 1.

Table 1 MIC determination results of dihydromyricetin and ceftiofur hydrochloride on ATCC 33591

combination drug MIC(μg/mL) FIC joint effect Dihydromyricetin 64 / / ceftiofur hydrochloride 128 / / Ceftiofur hydrochloride when adding 1/2 MIC dihydromyricetin 16 0.625 additive effect Ceftiofur hydrochloride when adding 1/4MIC dihydromyricetin 32 0.5 synergy Ceftiofur hydrochloride when adding 1/8MIC dihydromyricetin 64 0.625 additive effect

It can be seen from Table 1 that the MIC of ceftiofur hydrochloride to ATCC 33591 is 128 μg/mL, and the MIC of dihydromyricetin to ATCC 33591 is 64 μg/mL. When adding 1/2MIC(32μg/mL), 1/4MIC(16μg/mL), 1/8MIC(8μg/mL) dihydromyricetin, the MICs of ceftiofur hydrochloride were 16, 32, 64μg/mL The MIC values of ceftiofur hydrochloride decreased by 8, 4, and 2 times, respectively. As the concentration of dihydromyricetin decreased, the fold reduction of the MIC value of ceftiofur hydrochloride also decreased.

When the FIC in Table 1 is both 0.5, taking into account the reduction of antibiotic usage and bacterial resistance, a low-dose ceftiofur hydrochloride is selected, namely: 16 μg/mL dihydromyricetin (that is, 1/4 MIC concentration) in combination with ceftiofur hydrochloride, the MIC of ATCC 33591 decreased from 128 μg/mL to 32 μg/mL, the FIC was 0.5, and it had a synergistic antibacterial effect. The results showed that 16 μg/mL dihydromyricetin reversed the resistance of ATCC 33591 to ceftiofur hydrochloride.

Note:

When ceftiofur hydrochloride and 1/2MIC dihydromyricetin are used in combination, FIC=[32÷64+16÷128]=0.625;

When ceftiofur hydrochloride and 1/4MIC dihydromyricetin are used in combination, FIC=[16÷64+32÷128]=0.5;

When ceftiofur hydrochloride and 1/8MIC dihydromyricetin are used in combination, FIC=[8÷64+64÷128]=0.625;

The unit of drug dosage is μg/mL.

Combined application experiment for MRSA strain 1 of pig origin (healthy pig nasal cavity isolate 2AY40-2), the strain was provided by the Research Center of Traditional Chinese Veterinary Medicine of China Agricultural University.

The results of the combined drug susceptibility test are shown in Table 2.

Table 2 MIC determination results of dihydromyricetin and ceftiofur hydrochloride on pig-derived MRSA strain 1

combination drug MIC(μg/mL) FIC joint effect Dihydromyricetin 256 / / ceftiofur hydrochloride 64 / / Ceftiofur hydrochloride when adding 1/2 MIC dihydromyricetin 2 0.53125 additive effect Ceftiofur hydrochloride when adding 1/4MIC dihydromyricetin 2 0.28125 synergy Ceftiofur hydrochloride when adding 1/8MIC dihydromyricetin 2 0.15625 synergy Ceftiofur hydrochloride when adding 1/16MIC dihydromyricetin 4 0.125 synergy Ceftiofur hydrochloride when adding 1/32MIC dihydromyricetin 8 0.1875 synergy Ceftiofur hydrochloride when adding 1/64MIC dihydromyricetin 16 0.28125 synergy

It can be seen from Table 2 that the MIC of ceftiofur hydrochloride against MRSA strain 1 is 64 μg/mL, and the MIC of dihydromyricetin against MRSA strain 1 is 256 μg/mL. Add 1/2MIC(128μg/mL), 1/4MIC(64μg/mL), 1/8MIC(32μg/mL), 1/16MIC(16μg/mL), 1/32MIC(8μg/mL), 1/64MIC( 4μg/mL) dihydromyricetin, the MICs of ceftiofur hydrochloride were 0.053125, 0.28125, 0.15625, 0.125, 0.1875, 0.28125μg/mL, which decreased by 32, 32, and 32 compared with the MIC values of ceftiofur hydrochloride alone. , 16, 8, 4 times. As the concentration of dihydromyricetin decreased, the fold reduction of the MIC value of ceftiofur hydrochloride also decreased.

Available in Table 2: 16 μg/mL of dihydromyricetin (i.e., 1/16 MIC concentration) combined with ceftiofur hydrochloride reduces the MIC of MRSA strain 1 from 64 μg/mL to 4 μg/mL, the FIC is 0.125, and has Significant synergistic antibacterial effect. The results showed that 16 μg/mL (ie 1/16 MIC concentration) of dihydromyricetin reversed the resistance of MRSA strain 1 to ceftiofur hydrochloride.

Note:

When ceftiofur hydrochloride and 1/2MIC dihydromyricetin are used in combination, FIC=[128÷256+2÷64]=0.53125;

When ceftiofur hydrochloride and 1/4MIC dihydromyricetin are used in combination, FIC=[64÷256+2÷64]=0.28125;

When ceftiofur hydrochloride and 1/8MIC dihydromyricetin are used in combination, FIC=[32÷256+2÷64]=0.15625;

When ceftiofur hydrochloride and 1/16MIC dihydromyricetin are used in combination, FIC=[16÷256+4÷64]=0.125;

When ceftiofur hydrochloride and 1/32MIC dihydromyricetin are used in combination, FIC=[8÷256+8÷64]=0.1875;

When ceftiofur hydrochloride and 1/64MIC dihydromyricetin are used in combination, FIC=[16÷256+16÷64]=0.2812;

The unit of drug dosage is μg/mL.

Combined application experiment for MRSA strain 2 (healthy pig nasal isolate 2AY63), the strain was provided by the Research Center of Traditional Chinese Veterinary Medicine of China Agricultural University.

The test results are shown in Table 3.

Table 3 MIC determination results of dihydromyricetin and ceftiofur hydrochloride to MRSA strain 2

combination drug MIC(μg/mL) FIC joint effect Dihydromyricetin 128 / / ceftiofur hydrochloride 64 / / Ceftiofur hydrochloride when adding 1/2 MIC dihydromyricetin 2 0.53125 additive effect Ceftiofur hydrochloride when adding 1/4MIC dihydromyricetin 4 0.3125 synergy Ceftiofur hydrochloride when adding 1/8MIC dihydromyricetin 8 0.25 synergy Ceftiofur hydrochloride when adding 1/16MIC dihydromyricetin 16 0.3125 synergy Ceftiofur hydrochloride when adding 1/32MIC dihydromyricetin 32 0.53125 additive effect

It can be seen from Table 3 that the MIC of ceftiofur hydrochloride against MRSA strain 2 is 64 μg/mL, and the MIC of dihydromyricetin against MRSA strain 2 is 128 μg/mL. When adding 1/2MIC(64μg/mL), 1/4MIC(32μg/mL), 1/8MIC(16μg/mL), 1/16MIC(8μg/mL), 1/32MIC(4μg/mL) dihydromyricetin , the MIC of ceftiofur hydrochloride was 2, 4, 8, 16, and 32 μg/mL, which were 32, 16, 8, 4, and 2 times lower than the MIC value of ceftiofur hydrochloride alone. As the concentration of dihydromyricetin decreased, the fold reduction of the MIC value of ceftiofur hydrochloride also decreased.

Available in Table 3: The MIC of 16 μg/mL dihydromyricetin (that is, using 1/8 MIC concentration) and ceftiofur hydrochloride in combination with MRSA strain 2 decreased from 64 μg/mL to 8 μg/mL, FIC was 0.25, with Significant synergistic antibacterial effect. The results showed that 16 μg/mL (ie 1/8 MIC concentration) of dihydromyricetin reversed the resistance of MRSA strain 2 to ceftiofur hydrochloride.

Note:

When ceftiofur hydrochloride and 1/2MIC dihydromyricetin are used in combination, FIC=[64÷128+2÷64]=0.53125;

When ceftiofur hydrochloride and 1/4MIC dihydromyricetin are used in combination, FIC=[32÷128+4÷64]=0.3125;

When ceftiofur hydrochloride and 1/8MIC dihydromyricetin are used in combination, FIC=[16÷128+8÷64]=0.25;

When ceftiofur hydrochloride and 1/16MIC dihydromyricetin are used in combination, FIC=[8÷128+16÷64]=0.3125;

When ceftiofur hydrochloride and 1/32MIC dihydromyricetin are used in combination, FIC=[4÷128+32÷64]=0.53125;

The unit of drug dosage is μg/mL.

Combined application experiment for MRSA strain 3 (healthy pig nasal isolate 2AY61), the strain was provided by the Research Center of Traditional Chinese Veterinary Medicine of China Agricultural University.

The test results are shown in Table 4.

Table 4 MIC determination results of dihydromyricetin and ceftiofur hydrochloride to MRSA strain 3

combination drug MIC(μg/mL) FIC joint effect Dihydromyricetin 256 / / ceftiofur hydrochloride 64 / / Ceftiofur hydrochloride when adding 1/2 MIC dihydromyricetin 2 0.53125 additive effect Ceftiofur hydrochloride when adding 1/4MIC dihydromyricetin 4 0.3125 synergy Ceftiofur hydrochloride when adding 1/8MIC dihydromyricetin 8 0.25 synergy Ceftiofur hydrochloride when adding 1/16MIC dihydromyricetin 16 0.3125 synergy Ceftiofur hydrochloride when adding 1/32MIC dihydromyricetin 32 0.53125 additive effect

It can be seen from Table 4 that the MIC of ceftiofur hydrochloride against MRSA strain 3 is 64 μg/mL, and the MIC of dihydromyricetin against MRSA strain 3 is 256 μg/mL. When adding 1/2MIC(128μg/mL), 1/4MIC(64μg/mL), 1/8MIC(32μg/mL), 1/16MIC(16μg/mL), 1/32MIC(8μg/mL) dihydromyricetin , the MIC of ceftiofur hydrochloride was 2, 4, 8, 16, and 32 μg/mL, which were 32, 16, 8, 4, and 2 times lower than the MIC value of ceftiofur hydrochloride alone. As the concentration of dihydromyricetin decreased, the fold reduction of the MIC value of ceftiofur hydrochloride also decreased.

Available in Table 3: The MIC of 32 μg/mL dihydromyricetin (that is, using 1/8 MIC concentration) in combination with ceftiofur hydrochloride for MRSA strain 3 decreased from 64 μg/mL to 8 μg/mL, FIC was 0.25, with Significant synergistic antibacterial effect. The results showed that 32 μg/mL (ie 1/8 MIC concentration) of dihydromyricetin reversed the resistance of MRSA strain 3 to ceftiofur hydrochloride.

Note:

When ceftiofur hydrochloride and 1/2MIC dihydromyricetin are used in combination, FIC=[128÷256+2÷64]=0.53125;

When ceftiofur hydrochloride and 1/4MIC dihydromyricetin are used in combination, FIC=[64÷256+4÷64]=0.3125;

When ceftiofur hydrochloride and 1/8MIC dihydromyricetin are used in combination, FIC=[32÷256+8÷64]=0.25;

When ceftiofur hydrochloride and 1/16MIC dihydromyricetin are used in combination, FIC=[16÷256+16÷64]=0.3125;

When ceftiofur hydrochloride and 1/32MIC dihydromyricetin are used in combination, FIC=[8÷256+32÷64]=0.53125;

The unit of drug dosage is μg/mL.

Combined application experiments for MRSA strain 4 (healthy pig nasal isolate 2AY47), the strain was provided by the Research Center of Chinese Veterinary Medicine, China Agricultural University.

The test results are shown in Table 5.

Table 5 MIC determination results of dihydromyricetin and ceftiofur hydrochloride to MRSA strain 4

combination drug MIC(μg/mL) FIC joint effect Dihydromyricetin 128 / / ceftiofur hydrochloride 16 / / Ceftiofur hydrochloride when adding 1/2 MIC dihydromyricetin 2 0.625 additive effect Ceftiofur hydrochloride when adding 1/4MIC dihydromyricetin 4 0.5 synergy Ceftiofur hydrochloride when adding 1/8MIC dihydromyricetin 8 0.625 additive effect

It can be seen from Table 5 that the MIC of ceftiofur hydrochloride against MRSA strain 4 is 16 μg/mL, and the MIC of dihydromyricetin against MRSA strain 4 is 128 μg/mL. When adding 1/2MIC(64μg/mL), 1/4MIC(32μg/mL), 1/8MIC(16μg/mL) dihydromyricetin, the MICs of ceftiofur hydrochloride were 2, 4, and 8μg/mL, which were higher than those of single The MIC values of ceftiofur hydrochloride decreased by 8, 4, and 2 times, respectively. As the concentration of dihydromyricetin decreased, the fold reduction of the MIC value of ceftiofur hydrochloride also decreased.

Available in Table 3: The MIC of 32 μg/mL dihydromyricetin (that is, using 1/4 MIC concentration) combined with ceftiofur hydrochloride against MRSA strain 4 decreased from 16 μg/mL to 4 μg/mL, FIC was 0.5, with Significant synergistic antibacterial effect. The results showed that 32 μg/mL (ie 1/8 MIC concentration) of dihydromyricetin reversed the resistance of MRSA strain 4 to ceftiofur hydrochloride.

Note:

When ceftiofur hydrochloride and 1/2MIC dihydromyricetin are used in combination, FIC=[64÷128+2÷16]=0.625;

When ceftiofur hydrochloride and 1/4MIC dihydromyricetin are used in combination, FIC=[32÷128+4÷16]=0.5;

When ceftiofur hydrochloride and 1/8MIC dihydromyricetin are used in combination, FIC=[16÷128+8÷16]=0.625;

The unit of drug dosage is μg/mL.

Combined application experiment for MRSA strain 5 (healthy pig nasal isolate 2AY44), the strain was provided by the Research Center of Traditional Chinese Veterinary Medicine of China Agricultural University.

The test results are shown in Table 6.

Table 6 MIC determination results of dihydromyricetin and ceftiofur hydrochloride to MRSA strain 5

combination drug MIC(μg/mL) FIC joint effect Dihydromyricetin 128 / / ceftiofur hydrochloride 32 / / Ceftiofur hydrochloride when adding 1/2 MIC dihydromyricetin 2 0.5625 additive effect Ceftiofur hydrochloride when adding 1/4MIC dihydromyricetin 4 0.375 synergy Ceftiofur hydrochloride when adding 1/8MIC dihydromyricetin 8 0.375 synergy Ceftiofur hydrochloride when adding 1/16MIC dihydromyricetin 16 0.5625 additive effect

It can be seen from Table 6 that the MIC of ceftiofur hydrochloride against MRSA strain 5 is 32 μg/mL, and the MIC of dihydromyricetin against MRSA clinical strain 5 is 128 μg/mL. When adding 1/2MIC(64μg/mL), 1/4MIC(32μg/mL), 1/8MIC(16μg/mL), 1/16MIC(8μg/mL) dihydromyricetin, the MIC of ceftiofur hydrochloride is 2 , 4, 8, and 16 μg/mL, the MIC values of ceftiofur hydrochloride alone decreased by 16, 8, 4, and 2 times, respectively. As the concentration of dihydromyricetin decreased, the fold reduction of the MIC value of ceftiofur hydrochloride also decreased.

Available in Table 3: 32 μg/mL of dihydromyricetin (i.e., 1/4 MIC concentration) combined with ceftiofur hydrochloride has MIC for MRSA clinical strain 5 decreased from 32 μg/mL to 4 μg/mL, FIC is 0.375, It has obvious synergistic antibacterial effect. The results showed that 32 μg/mL (ie 1/4MIC concentration) of dihydromyricetin reversed the resistance of MRSA strain 5 to ceftiofur hydrochloride.

Note:

When ceftiofur hydrochloride and 1/2MIC dihydromyricetin are used in combination, FIC=[64÷128+2÷32]=0.5625;

When ceftiofur hydrochloride and 1/4MIC dihydromyricetin are used in combination, FIC=[32÷128+4÷32]=0.375;

When ceftiofur hydrochloride and 1/8MIC dihydromyricetin are used in combination, FIC=[16÷128+8÷32]=0.375;

When ceftiofur hydrochloride and 1/16MIC dihydromyricetin are used in combination, FIC=[8÷128+16÷32]=0.5625;

The unit of drug dosage is μg/mL.

Detailed ways

The following examples further illustrate the present invention, but are not intended to limit the present invention.

Each dose of dihydromyricetin is approximately 2 g, and each dose of ceftiofur hydrochloride is approximately 1 g.

Example 1

Preparation of ceftiofur hydrochloride pharmaceutical preparation:

1 g of ceftiofur hydrochloride was dissolved in 100 mL of polyethylene glycol for injection, and after aseptic processing, it was used as an injection.

Preparation of dihydromyricetin pharmaceutical preparation:

Dihydromyricetin 2g is aseptically packaged in powder form.

Combination Packaging:

Pack ceftiofur hydrochloride injection and dihydromyricetin powder into their respective medicines, and then put the two medicines together into the same large packaging box. Before use, ceftiofur hydrochloride injection and dihydromyricetin powder are brewed. .

Example 2

Preparation of ceftiofur hydrochloride pharmaceutical preparation:

1 g of ceftiofur hydrochloride was dissolved in 100 mL of polyethylene glycol for injection, and after aseptic processing, it was used as an injection.

Preparation of dihydromyricetin pharmaceutical preparation:

Dihydromyricetin 5g is aseptically packaged in powder form.

Combination Packaging:

Pack ceftiofur hydrochloride injection and dihydromyricetin powder into their respective medicines, and then put the two medicines together into the same large packaging box. Before use, ceftiofur hydrochloride injection and dihydromyricetin powder are brewed. .

Example 3

Preparation of ceftiofur hydrochloride pharmaceutical preparation:

1 g of ceftiofur hydrochloride was dissolved in 100 mL of polyethylene glycol for injection, and after aseptic processing, it was used as an injection.

Preparation of dihydromyricetin pharmaceutical preparation:

Dihydromyricetin 8g is aseptically packaged in powder form.

Combination Packaging:

Pack ceftiofur hydrochloride injection and dihydromyricetin powder into their respective medicines, and then put the two medicines together into the same large packaging box. Before use, ceftiofur hydrochloride injection and dihydromyricetin powder are brewed. .

Example 4

Preparation of ceftiofur hydrochloride pharmaceutical preparation:

1 g of ceftiofur hydrochloride was dissolved in 200 mL of polyethylene glycol for injection, and after aseptic processing, it was used as an injection.

Preparation of dihydromyricetin pharmaceutical preparation:

Dihydromyricetin 10g is aseptically packaged in powder form.

Combination Packaging:

Pack ceftiofur hydrochloride injection and dihydromyricetin powder into their respective medicines, and then put the two medicines together into the same large packaging box. Before use, ceftiofur hydrochloride injection and dihydromyricetin powder are brewed. .

Claims (10)

1. A pharmaceutical composition for resisting MRSA drug-resistant bacteria infection is a combination of two drugs, namely dihydromyricetin and ceftiofur hydrochloride.

2. The pharmaceutical combination according to claim 1, wherein the ratio of dihydromyricetin to ceftiofur hydrochloride in the pharmaceutical combination is 1-10: 1.

3. The pharmaceutical combination according to claim 2, wherein: in the medicine composition, the weight ratio of dihydromyricetin to ceftiofur hydrochloride is 1-5: 1.

4. The pharmaceutical combination according to claim 3, wherein: in the medicine composition, the weight ratio of dihydromyricetin to ceftiofur hydrochloride is 1-2: 1.

5. The pharmaceutical combination according to claim 4, wherein: in the medicine composition, the weight ratio of dihydromyricetin to ceftiofur hydrochloride is 2: 1.

6. The pharmaceutical combination according to claim 1, characterized in that: the combination comprises a pharmaceutical composition prepared by mixing dihydromyricetin and ceftiofur hydrochloride, and is applied simultaneously when in use, or the dihydromyricetin and the ceftiofur hydrochloride are respectively prepared into the pharmaceutical composition and are used together when in use.

7. The pharmaceutical combination according to claim 1, characterized in that: the pharmaceutical composition prepared by mixing dihydromyricetin and ceftiofur hydrochloride can also comprise other pharmaceutical carriers or auxiliary material components, and the dihydromyricetin and the ceftiofur hydrochloride are respectively prepared into pharmaceutical compositions, and are respectively packaged and used in a combined way when being used.

8. The pharmaceutical combination according to claim 7, wherein: the pharmaceutical composition is in the form of a pharmaceutical preparation and is selected from oral preparations and non-oral preparations, wherein the non-oral preparations comprise injections.

9. Application of dihydromyricetin in preparing medicine for increasing resistance of cephalosporin antibiotics to MRSA drug-resistant bacteria.

10. The use according to claim 9, wherein the cephalosporin antibiotic is ceftiofur hydrochloride and the dihydromyricetin and ceftiofur hydrochloride have a synergistic effect when the weight ratio of dihydromyricetin to ceftiofur hydrochloride is 2: 1.